PROJECT SUMMARY/ABSTRACT In this proposal, I aim to develop natural materials-based hydrogel cardiac tissue mimics that can serve as in vitro test beds for congenital heart disease (CHD). The motivation for this work is the lack of three- dimensional in vitro models for CHD that currently exist, and the little information known about the biophysical inputs of disease progression. CHD affects 1 percent of all live births worldwide. Numerous genetic mutations resulting in cardiac maldevelopment exist, a common one of which is a mutation in the Nkx2-5 gene. Preliminary data in an Nkx2-5 mutant mouse model from collaborator Dr. Kasahara revealed that approximately 20 genes are downregulated in the ventricles of diseased versus healthy hearts, two thirds of which are localized to the cell membrane or extracellular space. As the extracellular matrix (ECM) has been shown to play a role in many cellular processes such as migration, differentiation, and proliferation, this data warrants further exploration. Additionally, mutant hearts were shown to have ventricular noncompaction, a phenotype in which the heart?s trabecular layer is spongy rather than firm, limiting the heart?s capacity to pump blood effectively. Hence, the overall hypothesis for this project is that the Nkx2-5 mutation influences the biophysical properties of embryonic cardiac tissue, which in turn dynamically regulate developing cardiac cells. Replicating this disease phenotype in vitro could provide an effective method for studying the mechanisms of cardiac maldevelopment. An ECM component of interest in this project is hyaluronic acid (HA) because it is found ubiquitously throughout the ECM of the body, including cardiac tissue, and provides an excellent base for the development of hydrogel tissue mimics. The Schmidt lab has engineered HA-based hydrogels to mimic the mechanical properties of various ECM landscapes, independent of composition. These techniques will be applied to the creation of hydrogel scaffolds in this project. To this end, I propose 3 aims to accomplish the tasks of creating my ECM-based hydrogel tissue mimics. In Aim 1, I will determine the biophysical properties of cardiac tissue affected by the Nkx2-5 mutation. To do this I will assess ECM composition of healthy and diseased hearts through tandem mass spectrometry and Western blotting, and mechanical properties through indentation. In Aim 2, I will develop ECM-based hydrogel scaffolds to mimic the biophysical properties of healthy and diseased cardiac tissues and establish these scaffolds as 3D cell culture systems. To accomplish this, I will create HA- based hydrogel scaffolds with ECM compositions and mechanical properties matched to those of diseased and healthy heart tissue. In Aim 3, I will evaluate the biophysical properties that modify the fate of developing myocardial cells. For these studies, cardiomyocytes isolated from healthy and diseased hearts will be cultured in mutant and wild type test beds. The assumption of cell noncompaction phenotype as well as gene and protein expression will be assessed, and hydrogels will be indented to assess potential mechanical remodeling by cells.